本文選題：γ-Fe_2O_3 + 雜交��； 參考：《湖南工業(yè)大學(xué)》2012年碩士論文

【摘要】：聚合酶鏈?zhǔn)椒磻?yīng)（polymerase chain reaction, PCR）由于其特異性強(qiáng)、靈敏度高、快速簡(jiǎn)便等優(yōu)點(diǎn)，一直是生命科學(xué)領(lǐng)域一種重要的研究手段。增強(qiáng)其特異性，提高其靈敏度，是PCR技術(shù)研究的重點(diǎn)。 我們制備了平均粒徑為15nm的γ-Fe_2O_3磁性納米粒子，并對(duì)其進(jìn)行鏈霉親和素修飾。實(shí)驗(yàn)證明鏈霉親和素修飾的γ-Fe_2O_3磁性納米粒子具有良好的生物活性，可以進(jìn)一步用于生物檢測(cè)。 我們將磁性納米粒子γ-Fe_2O_3和多重PCR技術(shù)相結(jié)合，進(jìn)行了磁富集多重PCR擴(kuò)增，以增強(qiáng)其特異性，提高檢測(cè)靈敏度。 首先我們對(duì)該方法的可行性進(jìn)行分析，進(jìn)行了磁富集靶序列單重PCR擴(kuò)增。在這一實(shí)驗(yàn)過(guò)程中，先將生物素修飾的特異性引物和靶序列雜交，然后通過(guò)鏈霉親和素修飾的磁性納米粒子γ-Fe_2O_3將其富集到表面，通過(guò)變性獲取單鏈靶序列，再使用目的序列的擴(kuò)增引物進(jìn)行PCR擴(kuò)增。通過(guò)對(duì)實(shí)驗(yàn)條件的優(yōu)化，，我們得到靶序列和特異性引物雜交的最適溫度為52.5℃，鏈霉親和素修飾的磁性納米粒子γ-Fe_2O_3的最佳用量為90μg，該方法對(duì)靶序列檢測(cè)的靈敏度為5×10-10ng/μL。確定了該方法的可行性。 然后我們?cè)O(shè)計(jì)了AGT基因M235T位點(diǎn)和A-6G位點(diǎn)，MTHFR基因A1298C位點(diǎn)和C677T位點(diǎn)的擴(kuò)增引物，在磁富集單重PCR的基礎(chǔ)之上，對(duì)這四個(gè)位點(diǎn)進(jìn)行磁富集多重PCR擴(kuò)增，并優(yōu)化了擴(kuò)增條件。通過(guò)優(yōu)化實(shí)驗(yàn)我們得到，這四個(gè)位點(diǎn)的磁富集多重PCR的退火溫度為56℃，在30μL擴(kuò)增體系中，25mmol/L的Mg2+用量為2.0μL，10μmol/L的dNTP用量為1.5μL，5U/μL的Taq酶用量為0.7μL，10mmol/L的四個(gè)位點(diǎn)引物的用量分別為：C677T1μL、M235T0.7μL、A1298C1μL、A-6G1.5μL。同時(shí)得到這四個(gè)位點(diǎn)的磁富集多重PCR擴(kuò)增的靈敏度為5×10-9ng/μL。 最后，結(jié)合通用引物PCR技術(shù)和基因芯片技術(shù)，對(duì)磁富集多重PCR擴(kuò)增方法在這四個(gè)位點(diǎn)的SNP分型上進(jìn)行了初步應(yīng)用研究。
[Abstract]:Polymerase chain reaction (PCR) has been an important research method in the field of life science because of its high specificity, high sensitivity, rapidity and simplicity. Enhancing its specificity and sensitivity is the focus of PCR technology. 緯 -Fe _ 2O _ 3 magnetic nanoparticles with average diameter of 15nm were prepared and modified by streptavidin. The results show that the 緯 -Fe _ 2O _ 3 magnetic nanoparticles modified by Streptomycin have good biological activity and can be used for biological detection. The magnetic nanoparticles 緯 -Fe2O3 and multiplex PCR were combined to amplify PCR with magnetic enrichment to enhance its specificity and detection sensitivity. Firstly, we analyzed the feasibility of this method, and carried out single PCR amplification of magnetic enrichment target sequence. In this experiment, the specific primers and target sequences modified by biotin were first hybridized and then enriched onto the surface by the magnetic nanoparticles 緯 -Fe2O3 modified by streptavidin, and the single-chain target sequences were obtained by denaturation. The primers of the target sequence were used for PCR amplification. By optimizing the experimental conditions, the optimum temperature of target sequence and specific primer hybridization was 52.5 鈩

本文編號(hào)：1855709